Compositions and methods for sirna inhibition of angiopoietin 1 and 2 and their receptor tie2

ABSTRACT

RNA interference using small interfering RNAs which are specific for mRNA produced from the Ang1, Ang2 or Tie2 genes inhibits expression of these genes. Diseases which involve Ang1, Ang2 or Tie2 mediated angiogenesis, such as inflammatory and autoimmune diseases, diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering the small interfering RNAs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 10/827,759, filed Apr. 19, 2004, which claims thebenefit of U.S. Provisional Application No. 60/463,981, filed on Apr.18, 2003; the entire contents of which is hereby incorporated byreference in its their entirety.

FIELD OF THE INVENTION

This invention relates to the regulation of angiopoietin 1, angiopoietin2 and Tie2 gene expression by small interfering RNA, in particular fortreating diseases or conditions involving angiogenesis.

BACKGROUND OF THE INVENTION

Angiogenesis or “neovascularization” is the formation of new bloodvessels from the endothelial cells (EC) of preexisting blood vessels.This process involves EC migration, proliferation, and differentiation,which begins with localized breakdown of the basement membrane in theparent vessel. The EC then migrate away from the parent vessel into theinterstitial extracellular matrix (ECM) to form a capillary sprout,which elongates due to continued migration and proliferation of thecells.

Angiogenesis is typically held under strict control, and under normalconditions occurs only under certain defined physiological processes.For example, angiogenesis occurs during embryogenesis, post-natalgrowth, wound repair, and menstruation. Uncontrolled angiogenesis,however, can result in pathogenic conditions where the developing bloodvessels destroy the surrounding tissue or sustain malignancies. Suchpathogenic conditions include diabetic retinopathy, psoriasis, exudativeor “wet” age-related macular degeneration (“AMD”), inflammatorydisorders, and most cancers. AMD in particular is a clinically importantangiogenic disease. This condition is characterized by choroidalneovascularization in one or both eyes in aging individuals, and is themajor cause of blindness in industrialized countries.

Two key regulators of angiogenesis are angiopoietin-1 (“Ang1”) andangiopoietin-2 (“Ang2”). These regulators can act in concert withvascular endothelial growth factor (“VEGF”) to regulate angiogenesis,although inhibition of Ang1 or Ang2 alone appears to blockneovascularization. Ang1, Ang2 and VEGF exert their effect on EC throughthe two VEGF receptors and another tyrosine kinase receptor called“tyrosine kinase with immunoglobulin and epidermal growth factorhomology domains 2” or “Tie2.” Hackett et al. (2002), J. Cell. Phys.192: 182-187. Whereas VEGF binding to its receptors is crucial forinitiating the angiogenic process, Ang1 and Ang2 bind to Tie2 andmodulate maturation of the new blood vessels. Ang1 and Ang2 are alsoinvolved in maintaining endothelial cell integrity. Lobov et al. (2002),Proc. Nat. Acad. Sci USA 99: 11205-11210. As discussed below, agentswhich bind to and block the Tie2 receptor can also inhibit angiogenesis.

Ang1 and Ang2 are differentially expressed, and early studies indicatedthat Ang1 promoted neovascularization and Ang2 was an angiogenesisantagonist. However, evidence now shows that Ang2 can increase bloodvessel diameter and promote remodeling of the basal lamina. Ang2 alsoappears to induce EC proliferation, migration and sprouting of bloodvessels in the presence of VEGF. Lobov et al., 2002, supra.

Ang1 reportedly promotes angiogenesis during embryonic development, inparticular through the modulation of endothelial-stromal cellcommunication and by regulating the maturation and stability of bloodvessels. Lin P et al., Proc. Nat. Acad. Sci. USA 95: 8829-8834 (1998).However, the widespread expression of Ang1 and Tie2 in vascularendothelium, and phosphorylation of Tie2 in quiescent adult vasculaturealso suggest that Ang1 is involved in postnatal angiogenesis. Takagi etal. (2003), Inv. Ophthalm. Vis. Sci. 44: 393-402.

In contrast to the more extensive expression patterns of Ang1 and Tie2,Ang2 appears to be expressed only at sites of vascular remodeling.Takagi et al. (2003), supra. For example, Ang2 expression is markedlyincreased in ovary, uterus and placenta during menstruation. Ang2expression levels also follow a cyclical pattern of expression in thecorpus luteum, which parallels the cycle of quiescence, angiogenesis andvascular regression of this structure (i.e., Ang2 levels are low duringquiescence and high during angiogenesis and regression). Hackett et al.,2002, supra. Ang2 is also induced by hypoxic cytokines, including VEGF,and is expressed in tissues undergoing pathologic angiogenesisassociated with tumors, AMD and in an animal model of retinal ischemia.Takagi et al., 2003, supra. Moreover, Ang2 is upregulated in theepiretinal membranes of patients with ischemic retinal disorders, butnot in membranes from patients with non-ischemic retinal disorders. Theexpression of Ang1, however, remains similar in epiretinal membranesfrom patients with ischemic or non-ischemic disorders. Takagi et al.,2003, supra.

Ang2 and Tie2 are co-localized in the EC of highly vascularized regions,and Tie2 is overexpressed in areas of vascular remodeling. Asahara T. etal., Circ. Res. 83: 223-240 report that Ang1 and Ang2 have similarsynergistic effects with VEGF to promote angiogenesis in a mouse cornealneovascularization assay. Thus, Ang1, Ang2 and Tie2 play an importantrole in both normal and pathogenic neovascularization in developing andadult organisms.

Ang1, Ang2 or Tie2 are therefore attractive therapeutic targets fortreatment of pathogenic angiogenesis. For example, Lin Pet al. (1998),supra, inhibited tumor growth and metastasis in a mouse model byexpressing a soluble recombinant Tie2 receptor. The recombinant Tie2protein blocked ligand binding to endogenous Tie2 receptors, but likelyproduced only a stoichiometric reduction in Ang2/Tie2 binding. Takagi etal., 2003, supra inhibited of Tie2 signaling with a soluble fusionprotein containing the ectoplasmic domain of Tie2, which suppressedhypoxia-induced retinal angiogenesis both in vitro and in vivo. Asaharaet al. (1998), supra showed that administration of a soluble Tie2receptor abolished the effects of Ang1 or Ang2 on VEGF-inducedneovascularization in the mouse cornea. However, therapeutic strategiesbased on agents such as soluble Tie2 receptors are not preferred,however, because such agents would likely be overwhelmed by the highproduction of Ang2 or Tie2 in the EC of highly vascularized areas.

RNA interference (hereinafter “RNAi”) is a method ofpost-transcriptional gene regulation that is conserved throughout manyeukaryotic organisms. RNAi is induced by short (i.e., <30 nucleotide)double stranded RNA (“dsRNA”) molecules which are present in the cell(Fire A et al. (1998), Nature 391: 806-811). These short dsRNAmolecules, called “short interfering RNA” or “siRNA,” cause thedestruction of messenger RNAs (“mRNAs”) which share sequence homologywith the siRNA to within one nucleotide resolution (Elbashir S M et al.(2001), Genes Dev, 15: 188-200). It is believed that the siRNA and thetargeted mRNA bind to an “RNA-induced silencing complex” or “RISC”,which cleaves the targeted mRNA. The siRNA is apparently recycled muchlike a multiple-turnover enzyme, with 1 siRNA molecule capable ofinducing cleavage of approximately 1000 mRNA molecules. siRNA-mediatedRNAi degradation of an mRNA is therefore more effective than currentlyavailable technologies for inhibiting expression of a target gene.

Elbashir S M et al. (2001), supra, has shown that synthetic siRNA of 21and 22 nucleotides in length, and which have short 3′ overhangs, areable to induce RNAi of target mRNA in a Drosophila cell lysate. Culturedmammalian cells also exhibit RNAi degradation with synthetic siRNA(Elbashir S M et al. (2001) Nature, 411: 494-498), and RNAi degradationinduced by synthetic siRNA has recently been shown in living mice(McCaffrey A P et al. (2002), Nature, 418: 38-39; Xia H et al. (2002),Nat. Biotech. 20: 1006-1010). The therapeutic potential of siRNA-inducedRNAi degradation has been demonstrated in several recent in vitrostudies, including the siRNA-directed inhibition of HIV-1 infection(Novina C D et al. (2002), Nat. Med. 8: 681-686) and reduction ofneurotoxic polyglutamine disease protein expression (Xia H et al.(2002), supra).

What is needed, therefore, are agents and methods which selectivelyinhibit expression of Ang1, Ang2 or Tie2 in catalytic orsub-stoichiometric amounts, in order to effectively decrease or blockangiogenesis.

SUMMARY OF THE INVENTION

The present invention is directed to siRNA which specifically target andcause RNAi-induced degradation of mRNA from Ang1, Ang2 or Tie2 genes.These siRNA degrade Ang1, Ang2 or Tie2 mRNA in substoichiometricamounts. The siRNA compounds and compositions of the invention are, thusused to inhibit angiogenesis. In particular, the siRNA of the inventionare useful for treating cancerous tumors and disorders related to ocularneovascularization, such as age-related macular degeneration anddiabetic retinopathy.

Thus, the invention provides an isolated siRNA which targets human Ang1,Ang2 or Tie2 mRNA, or an alternative splice form, mutant or cognatethereof. The siRNA comprises a sense RNA strand and an antisense RNAstrand which form an RNA duplex. The sense RNA strand comprises anucleotide sequence substantially identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in the target mRNA.

The invention also provides recombinant plasmids and viral vectors whichexpress the siRNA of the invention, as well as pharmaceuticalcompositions comprising the siRNA of the invention and apharmaceutically acceptable carrier.

The invention further provides a method of inhibiting expression ofhuman Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant orcognate thereof, comprising administering to a subject an effectiveamount of the siRNA of the invention such that the target mRNA isdegraded.

The invention further provides a method of inhibiting angiogenesis in asubject, comprising administering to a subject an effective amount of ansiRNA targeted to human Ang1, Ang2 or Tie2 mRNA, or an alternativesplice form, mutant or cognate thereof.

The invention further provides a method of treating an angiogenicdisease, comprising administering to a subject in need of such treatmentan effective amount of an siRNA targeted to human Ang1, Ang2 or Tie2mRNA, or an alternative splice form, mutant or cognate thereof, suchthat angiogenesis associated with the angiogenic disease is inhibited.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a histogram showing the silencing effect of siRNA candidates,as measured by the levels of human angiopoietin 2 (“hANG2”) protein ingrowth medium removed from tissue culture wells containing HEK-293 cellstransfected with: twelve different siRNA targeted to hANG2 mRNA(hANG2#1-hANG2#12); with control nonspecific siRNA targeted to enhancedgreen fluorescent protein (“EGFP”); or with transfection reagentcontaining no siRNA (“no”). hANG2 protein level is given in picograms ofprotein per milliliter of growth medium (pg/ml), as measured by hANG2ELISA at 48 hours post-transfection.

FIG. 2 is a histogram showing lack of cytotoxicity in HEK-293 cellstransfected with twelve different siRNA targeted to hANG2 mRNA(hANG2#1-hANG2#12). Control cells were transfected with nonspecificsiRNA targeted to enhanced green fluorescent protein mRNA (“EGFP”), orwith transfection reagent containing no siRNA (“no”). Cytotoxicity ismeasured as percent growth of cells treated with siRNA vs. cells treatedwith transfection reagent alone.

FIG. 3 is a histogram showing the silencing effect of increasing dosesof hANG2#2 and hANG2#3 on the level of hANG2 protein secreted by HEK-293cells. The HEK-293 cells were transfected with 1 nanomolar (“nM”), 5 nM,or 25 nM hANG2#2 or hANG2#3 siRNA. Control cells were transfected with25 nM nonspecific siRNA targeted to enhanced green fluorescent proteinmRNA (“EGFP”), or with transfection reagent containing no siRNA (“no”).hANG2 protein level is given in picograms of protein per milliliter ofgrowth medium (pg/ml), as measured by hANG2 ELISA at 48 hourspost-transfection.

FIG. 4 is a histogram showing lack of cytotoxicity in HEK-293 cellstransfected with increasing doses of hANG2#2 and hANG2#3 siRNA. Controlcells were transfected with nonspecific siRNA targeted to enhanced greenfluorescent protein mRNA (“EGFP”), or with transfection reagentcontaining no siRNA (“no”). Cytotoxicity is measured as percent growthof cells treated with siRNA vs. cells treated with transfection reagentalone.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, all nucleic acid sequences herein are givenin the 5′ to 3′ direction. Also, all deoxyribonucleotides in a nucleicacid sequence are represented by capital letters (e.g., deoxythymidineis “T”), and ribonucleotides in a nucleic acid sequence are representedby lower case letters (e.g., uridine is “u”).

Compositions and methods comprising siRNA targeted to Ang1, Ang2 andTie2 mRNA are advantageously used to inhibit angiogenesis, in particularfor the treatment of angiogenic disease. The siRNA of the invention arebelieved to cause the RNA1-mediated degradation of these mRNAs, so thatthe protein products of the Ang1, Ang2 or Tie2 genes are not produced orare produced in reduced amounts. Because Ang1, Ang2 and Tie2 areinvolved in angiogenesis, the siRNA-mediated degradation of Ang1, Ang2or Tie2 mRNA inhibits the angiogenic process.

As used herein, siRNA which is “targeted to the Ang1, Ang2 or Tie2 mRNA”means siRNA in which a first strand of the duplex is substantiallyidentical to the nucleotide sequence of a portion of the Ang1, Ang2 orTie2 mRNA sequence. It is understood that the second strand of the siRNAduplex is complementary to both the first strand of the siRNA duplex andto the same portion of the Ang1, Ang2 or Tie2 mRNA.

The invention therefore provides isolated siRNA comprising shortdouble-stranded RNA from about 17 nucleotides to about 29 nucleotides inlength, preferably from about 19 to about 25 nucleotides in length, thatare targeted to the target mRNA. The siRNA's comprise a sense RNA strandand a complementary antisense RNA strand annealed together by standardWatson-Crick base-pairing interactions (hereinafter “base-paired”). Asis described in more detail below, the sense strand comprises a nucleicacid sequence which is substantially identical to a target sequencecontained within the target mRNA.

As used herein, a nucleic acid sequence “substantially identical” to atarget sequence contained within the target mRNA is a nucleic acidsequence which is identical to the target sequence, or which differsfrom the target sequence by one or more nucleotides. Sense strands ofthe invention which comprise nucleic acid sequences substantiallyidentical to a target sequence are characterized in that siRNAcomprising such sense strands induce RNAi-mediated degradation of mRNAcontaining the target sequence. For example, an siRNA of the inventioncan comprise a sense strand comprise nucleic acid sequences which differfrom a target sequence by one, two or three or more nucleotides, as longas RNAi-mediated degradation of the target mRNA is induced by the siRNA.

The sense and antisense strands of the present siRNA can comprise twocomplementary, single-stranded RNA molecules or can comprise a singlemolecule in which two complementary portions are base-paired and arecovalently linked by a single-stranded “hairpin” area. Without wishingto be bound by any theory, it is believed that the hairpin area of thelatter type of siRNA molecule is cleaved intracellularly by the “Dicer”protein (or its equivalent) to form a siRNA of two individualbase-paired RNA molecules (see Tuschl, T. (2002), supra). As describedbelow, the siRNA can also contain alterations, substitutions ormodifications of one or more ribonucleotide bases. For example, thepresent siRNA can be altered, substituted or modified to contain one ormore deoxyribonucleotide bases.

As used herein, “isolated” means synthetic, or altered or removed fromthe natural state through human intervention. For example, an siRNAnaturally present in a living animal is not “isolated,” but a syntheticsiRNA, or an siRNA partially or completely separated from the coexistingmaterials of its natural state is “isolated.” An isolated siRNA canexist in substantially purified form, or can exist in a non-nativeenvironment such as, for example, a cell into which the siRNA has beendelivered. By way of example, siRNA which are produced inside a cell bynatural processes, but which are produced from an “isolated” precursormolecule, are themselves “isolated” molecules. Thus, an isolated dsRNAcan be introduced into a target cell, where it is processed by the Dicerprotein (or its equivalent) into isolated siRNA.

As used herein, “target mRNA” means human Ang1, Ang2 or Tie2 mRNA,mutant or alternative splice forms of human Ang1, Ang2 or Tie2 mRNA, ormRNA from cognate Ang1, Ang2 or Tie2 genes. The human Ang1, Ang2 andTie2 mRNA sequences are described in GenBank Record Accession Nos.AY124380, NM_(—)00147 and L06139, respectively, as the cDNA equivalents.The human Ang1, Ang2 and Tie2 mRNA sequences are given herein as SEQ IDNO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, as the cDNAequivalents. One skilled in the art would understand that the cDNAsequence is equivalent to the mRNA sequence, and can be used for thesame purpose herein; i.e., the generation of siRNA for inhibitingexpression of Ang1, Ang2, or Tie2.

As used herein, a gene or mRNA which is “cognate” to human Ang1, Ang2 orTie2 is a gene or mRNA from another mammalian species which ishomologous to human Ang1, Ang2 or Tie2. For example, the partialsequence of Ang1 mRNA for the domesticated dog (Canis familiaris) isdescribed as the cDNA equivalent in GenBank Record Accession No.AF345932, which is given herein as SEQ ID NO: 4. The Mus musculus(mouse) Ang2 mRNA is described as the cDNA equivalent in GenBank RecordAccession No. NM_(—)007426, which is given herein as SEQ ID NO. 5. TheMus musculus (mouse) and Rattus norvegicus (rat) Tie2 mRNA sequences aredescribed as the cDNA equivalents in GenBank Record Accession Nos.NM_(—)013690 and NW_(—)043856, respectively. The mouse and rat Tie2 mRNAsequences are given herein as SEQ ID NO. 6 and SEQ ID NO. 7,respectively.

Alternative splice forms of human Ang1, Ang2 and Tie2 are also known.See, e.g., GenBank Record Accession No. AY121504, which describes asplice variant of human Ang1 as the cDNA equivalent (SEQ ID NO: 8). Kimet al., J. Biol. Chem. 275 (24), 18550-18556 (2000) and GenBank RecordAccession No. AF187858 describe an Ang2 splice variant encoding an Ang2protein lacking amino acids 96-148, given as the cDNA equivalent (SEQ 1DNO: 9). See also GenBank Record Accession No. AB086825, which describesa splice variant of Tie2 encoding a Tie2 protein lacking the epidermalgrowth factor-like domain, given as the cDNA equivalent (SEQ ID NO: 10).

The mRNA transcribed from the human Ang1, Ang2 or Tie2 genes can also beanalyzed for alternative splice forms using techniques well-known in theart. Such techniques include reverse transcription-polymerase chainreaction (RT-PCR), northern blotting and in-situ hybridization.Techniques for analyzing mRNA sequences are described, for example, inBusting S A (2000), J. Mol. Endocrinol. 25: 169-193, the entiredisclosure of which is herein incorporated by reference. Representativetechniques for identifying alternatively spliced mRNAs are alsodescribed below.

For example, databases that contain nucleotide sequences related to agiven disease gene can be used to identify alternatively spliced mRNA.Such databases include GenBank, Embase, and the Cancer Genome AnatomyProject (CGAP) database. The CGAP database, for example, containsexpressed sequence tags (ESTs) from various types of human cancers. AnmRNA or gene sequence from the Ang1, Ang2 or Tie2 genes can be used toquery such a database to determine whether ESTs representingalternatively spliced mRNAs have been found.

A technique called “RNAse protection” can also be used to identifyalternatively spliced Ang1, Ang2 or Tie2 mRNAs. RNAse protectioninvolves translation of a gene sequence into synthetic RNA, which ishybridized to RNA derived from other cells; for example, cells which areinduced to express Ang1, Ang2 or Tie2. The hybridized RNA is thenincubated with enzymes that recognize RNA:RNA hybrid mismatches. Smallerthan expected fragments indicate the presence of alternatively splicedmRNAs. The putative alternatively spliced mRNAs can be cloned andsequenced by methods well known to those skilled in the art.

RT-PCR can also be used to identify alternatively spliced Ang1, Ang2 orTie2 mRNAs. In RT-PCR, mRNA from vascular endothelial cells or cellsfrom other tissue known to express Ang1, Ang2 or Tie2 is converted intocDNA by the enzyme reverse transcriptase, using methods within the skillin the art. The entire coding sequence of the cDNA is then amplified viaPCR using a forward primer located in the 3′ untranslated region, and areverse primer located in the 5′ untranslated region. The amplifiedproducts can be analyzed for alternative splice forms, for example bycomparing the size of the amplified products with the size of theexpected product from normally spliced mRNA, e.g., by agarose gelelectrophoresis. Any change in the size of the amplified product canindicate alternative splicing.

The mRNA produced from mutant Ang1, Ang2 or Tie2 genes can also bereadily identified with the techniques described above for identifyingAng1, Ang2 or Tie2 alternative splice forms. As used herein, “mutant”Ang1, Ang2 or Tie2 genes or mRNA include human Ang1, Ang2 or Tie2 genesor mRNA which differ in sequence from the Ang1, Ang2 and Tie2 sequencesset forth herein. Thus, allelic forms of the Ang1, Ang2 or Tie2 genes,and the mRNA produced from them, are considered “mutants” for purposesof this invention. See also WO 02/20734, which describes several mutantsof Tie2, one of which is described in GenBank Record Accession No.AX398356, which is given herein as the cDNA equivalent in SEQ ID NO: 11.

It is understood that human Ang1, Ang2 or Tie2 mRNA may contain targetsequences in common with its respective alternative splice forms,cognates or mutants. A single siRNA comprising such a common targetingsequence can therefore induce RNAi-mediated degradation of thosedifferent mRNAs which contain the common targeting sequence.

The siRNA of the invention can comprise partially purified RNA,substantially pure RNA, synthetic RNA, or recombinantly produced RNA, aswell as altered RNA that differs from naturally-occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA; modifications that make the siRNA resistant tonuclease digestion (e.g., the use of 2′-substituted ribonucleotides ormodifications to the sugar-phosphate backbone); or the substitution ofone or more nucleotides in the siRNA with deoxyribonucleotides. siRNAwhich are exposed to serum, lachrymal fluid or other nuclease-richenvironments, or which are delivered topically (e.g., by eyedropper),are preferably altered to increase their resistance to nucleasedegradation. For example, siRNA which are administered intravascularlyor topically to the eye can comprise one or more phosphorothioatelinkages.

One or both strands of the siRNA of the invention can also comprise a 3′overhang. As used herein, a “3′ overhang” refers to at least oneunpaired nucleotide extending from the 3′-end of an RNA strand.

Thus in one embodiment, the siRNA of the invention comprises at leastone 3′ overhang of from 1 to about 6 nucleotides (which includesribonucleotides or deoxynucleotides) in length, preferably from 1 toabout 5 nucleotides in length, more preferably from 1 to about 4nucleotides in length, and particularly preferably from about 2 to about4 nucleotides in length.

In the embodiment in which both strands of the siRNA molecule comprise a3′ overhang, the length of the overhangs can be the same or differentfor each strand. In a most preferred embodiment, the 3′ overhang ispresent on both strands of the siRNA, and is 2 nucleotides in length.For example, each strand of the siRNA of the invention can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

In order to enhance the stability of the present siRNA, the 3′ overhangscan be also stabilized against degradation. In one embodiment, theoverhangs are stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, istolerated and does not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′-hydroxyl in the 2′-deoxythymidinesignificantly enhances the nuclease resistance of the 3′ overhang intissue culture medium.

In certain embodiments, the siRNA of the invention comprises thesequence AA(N19)TT or NA(N21), where N is any nucleotide. These siRNAcomprise approximately 30-70% GC, and preferably comprise approximately50% G/C. The sequence of the sense siRNA strand corresponds to (N19)TTor N21 (i.e., positions 3 to 23), respectively. In the latter case, the3′ end of the sense siRNA is converted to TT. The rationale for thissequence conversion is to generate a symmetric duplex with respect tothe sequence composition of the sense and antisense strand 3′ overhangs.The antisense RNA strand is then synthesized as the complement topositions 1 to 21 of the sense strand.

Because position 1 of the 23-nucleotide sense strand in theseembodiments is not recognized in a sequence-specific manner by theantisense strand, the 3′-most nucleotide residue of the antisense strandcan be chosen deliberately. However, the penultimate nucleotide of theantisense strand (complementary to position 2 of the 23-nucleotide sensestrand in either embodiment) is generally complementary to the targetedsequence.

In another embodiment, the siRNA of the invention comprises the sequenceNAR(N17)YNN, where R is a purine (e.g., A or G) and Y is a pyrimidine(e.g., C or u/T). The respective 21-nucleotide sense and antisense RNAstrands of this embodiment therefore generally begin with a purinenucleotide. Such siRNA can be expressed from pol III expression vectorswithout a change in targeting site, as expression of RNAs from pol IIIpromoters is only believed to be efficient when the first transcribednucleotide is a purine.

The siRNA of the invention can be targeted to any stretch ofapproximately 19-25 contiguous nucleotides in any of the target mRNAsequences (the “target sequence”). Techniques for selecting targetsequences for siRNA's are given, for example, in Tuschl T et al., “ThesiRNA User Guide,” revised Oct. 11, 2002, the entire disclosure of whichis herein incorporated by reference. “The siRNA User Guide” is availableon the world wide web at a website maintained by Dr. Thomas Tuschl,Department of Cellular Biochemistry, AG 105, Max-Planck-Institute forBiophysical Chemistry, 37077 Göttingen, Germany, and can be found byaccessing the website of the Max Planck Institute and searching with thekeyword “siRNA.” Thus, the sense strand of the present siRNA comprises anucleotide sequence substantially identical to any contiguous stretch ofabout 19 to about 25 nucleotides in the target mRNA.

Generally, a target sequence on the target mRNA can be selected from agiven cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′ direction) from thestart codon. The target sequence can, however, be located in the 5′ or3′ untranslated regions, or in the region nearby the start codon. Forexample, a suitable target sequence in the human Ang2 cDNA sequence is:

(SEQ ID NO: 12) AATGCTGTGCAGAGGGACGCG

Thus, an siRNA of the invention targeting SEQ ID NO: 12, and which has3′ uu overhangs on each strand (overhangs shown in bold), is:

(SEQ ID NO: 13) 5′-tgctgtgcagagggacgcguu-3′ (SEQ ID NO: 14)3′-uuucgacacgucucccugcgc-5′

An siRNA of the invention targeting SEQ ID NO: 12, but having 3′ TToverhangs on each strand (overhangs shown in bold) is:

(SEQ ID NO: 15) 5′-tgctgtgcagagggacgcgTT-3′ (SEQ ID NO: 16)3′-TTucgacacgucucccugcgc-5′

Another target sequence from the human Ang2 cDNA sequence is:

(SEQ ID NO: 17) AAGTATTAAATCAGACCACGA

Thus, an siRNA of the invention targeting SEQ ID NO: 17, and which has3′ uu overhangs on each strand (overhangs shown in bold), is:

(SEQ ID NO: 18) 5′-gtattaaatcagaccacgauu-3′ (SEQ ID NO: 19)3′-uucauaauuuagucuggugcu-5′

An siRNA of the invention targeting SEQ ID NO: 17, but having 3′ TToverhangs on each strand (overhangs shown in bold) is:

(SEQ ID NO: 20) 5′-gtattaaatcagaccacgaTT-3′ (SEQ ID NO: 21)3′-TTcauaauuuagucuggugcu-5′

A suitable target sequence in the human Ang1 cDNA sequence is:

(SEQ ID NO: 22) AATGCAGTTCAGAACCACACG

Thus, an siRNA of the invention targeting SEQ ID NO: 22, and which has3′ uu overhangs on each strand (overhangs shown in bold), is:

(SEQ ID NO: 23) 5′-tgcagttcagaaccacacguu-3′ (SEQ ID NO: 24)3′-uu acgucaagucuuggugugc-5′

An siRNA of the invention targeting SEQ ID NO: 22, but having 3′ TToverhangs on each strand (overhangs shown in bold) is:

(SEQ ID NO: 25) 5′-tgcagttcagaaccacacgTT-3′ (SEQ ID NO: 26)3′-TTacgucaagucuuggugugc-5′

Another target sequence from the human Ang1 cDNA is:

(SEQ ID NO: 27) AACTTCTCGACTTGAGATACA

An siRNA of the invention targeting SEQ ID NO: 27, but having 3′ uuoverhangs on each strand (overhangs shown in bold) is:

(SEQ ID NO: 28) 5′-cttctcgacttgagatacauu-3′ (SEQ ID NO: 29)3′-uugaagagcugaacucuaugu-5′

An siRNA of the invention targeting SEQ ID NO: 27, but having 3′ TToverhangs on each strand (overhangs shown in bold) is:

(SEQ ID NO: 30) 5′-cttctcgacttgagatacaTT-3′ (SEQ ID NO: 31)3′-TTgaagagcugaacucuaugu-5′

Other Ang1, Ang2 and Tie2 target sequences, from which siRNA of theinvention can be derived, include those given herein: Suitable humanAng1 target sequences include those of SEQ ID NOS; 32-227; suitablehuman Ang2 target sequences include those of SEQ ID NOS: 228-427; andsuitable human Tie2 target sequences include those of SEQ ID NOS:428-739. It is understood that the target sequences given herein arewith reference to the human Ang1, Ang2 or Tie2 cDNA, and thus thesesequences contain deoxythymidines represented by “T.” One skilled in theart would understand that, in the actual target sequence of the mRNA,the deoxythymidines would be replaced by uridines (“u”). Likewise, atarget sequence contained within an siRNA of the invention would alsocontain uridines in place of deoxythymidines.

The siRNA of the invention can be obtained using a number of techniquesknown to those of skill in the art. For example, the siRNA, can bechemically synthesized or recombinantly produced using methods known inthe art, such as the. Drosophila in vitro system described in U.S.published application 2002/0086356 of Tuschl et al., the entiredisclosure of which is herein incorporated by reference.

Preferably, the siRNA of the invention are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. The siRNA can be synthesized as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).

Alternatively, siRNA can also be expressed from recombinant circular orlinear DNA plasmids using any suitable promoter. Suitable promoters forexpressing siRNA of the invention from a plasmid include, for example,the U6 or H1 RNA pol III promoter sequences and the cytomegaloviruspromoter. Selection of other suitable promoters is within the skill inthe art. The recombinant plasmids of the invention can also compriseinducible or regulatable promoters for expression of the siRNA in aparticular tissue or in a particular intracellular environment.

The siRNA expressed from recombinant plasmids can either be isolatedfrom cultured cell expression systems by standard techniques, or can beexpressed intracellularly. The use of recombinant plasmids to deliversiRNA of the invention to cells in vivo is discussed in more detailbelow.

siRNA of the invention can be expressed from a recombinant plasmideither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Selection of plasmids suitable for expressing siRNA of the invention,methods for inserting nucleic acid sequences for expressing the siRNAinto the plasmid, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for exampleTuschl, T. (2002), Nat. Biotechnol, 20: 446-448; Brummelkamp T R et al.(2002), Science 296: 550-553; Miyagishi M et al. (2002), Nat.Biotechnol. 20: 497-500; Paddison P J et al. (2002), Genes Dev. 16:948-958; Lee N S et al. (2002), Nat. Biotechnol. 20: 500-505; and Paul CP et al. (2002), Nat. Biotechnol. 20: 505-508, the entire disclosures ofwhich are herein incorporated by reference.

In one embodiment, a plasmid expressing an siRNA of the inventioncomprises a sense RNA strand coding sequence in operable connection witha polyT termination sequence under the control of a human U6 RNApromoter, and an antisense RNA strand coding sequence in operableconnection with a polyT termination sequence under the control of ahuman U6 RNA promoter. Such a plasmid can be used in producing anrecombinant adeno-associated viral vector for expressing an siRNA of theinvention.

As used herein, “in operable connection with a polyT terminationsequence” means that the nucleic acid sequences encoding the sense orantisense strands are immediately adjacent to the polyT terminationsignal in the 5′ direction. During transcription of the sense orantisense sequences from the plasmid, the polyT termination signals actto terminate transcription.

As used herein, “under the control” of a promoter means that the nucleicacid sequences encoding the sense or antisense strands are located 3′ ofthe promoter, so that the promoter can initiate transcription of thesense or antisense coding sequences.

The siRNA of the invention can also be expressed from recombinant viralvectors intracellularly in vivo. The recombinant viral vectors of theinvention comprise sequences encoding the siRNA of the invention and anysuitable promoter for expressing the siRNA sequences. Suitable promotersinclude, for example, the U6 or HI RNA pol III promoter sequences andthe cytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the siRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver siRNA of the invention to cells in vivo is discussed in moredetail below.

siRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the siRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the siRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the siRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the siRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol., 70:520-532; Samulski R et al. (1989). J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference.

The ability of an siRNA containing a given target sequence to causeRNAi-mediated degradation of the target mRNA can be evaluated usingstandard techniques for measuring the levels of RNA or protein in cells.For example, siRNA of the invention can be delivered to cultured cells,and the levels of target mRNA can be measured by Northern blot or dotblotting techniques, or by quantitative RT-PCR. Alternatively, thelevels of Ang1, Ang2 or Tie2 protein in the cultured cells can bemeasured by ELISA or Western blot. Suitable protocols for the deliveryof siRNA to cultured cells, and assays for detecting protein and mRNAlevels in cultured cells, are given in the Examples below.

For example, cells which naturally express Ang1, Ang2 or Tie2, or whichare induced to express Ang1, Ang2 or Tie2, are grown to confluence insuitable cell culture vessels; e.g., 12- or 25-well culture plates or96-well microtiter plates. siRNA of the invention can be administered toone group of Ang1, Ang2 or Tie2 expressing cells. A non-specific siRNA(or no siRNA) can be administered to a second group of Ang1, Ang2 orTie2 expressing cells as a control. The cells are washed and directlyfixed to the microtiter plate wells with 1 to 2% paraformaldehyde.Nonspecific binding sites on the microtiter plate are blocked with 2%bovine serum albumin, and the cells incubated with an Ang1, Ang2 or Tie2specific monoclonal antibody. Bound Ang1, Ang2 or Tie2 antibody can bedetected, for example, by incubation with a 1:1000 dilution ofbiotinylated goat anti-mouse IgG (Bethesda Research Laboratories,Gaithersberg, Md.) for 1 hour at 37° C. and with a 1:1000 dilution ofstreptavidin conjugated to beta-galactosidase (Bethesda ResearchLaboratories) for 1 hour at 37° C. The amount of beta-galactosidasebound to the Ang1, Ang2 or Tie2 specific monoclonal antibody isdetermined, for example, by developing the microtiter plate in asolution of 3.3 mM chlorophenol red-beta-D-galactopyranoside, 50 mMsodium phosphate, 1.5 mM MgCl₂; pH 7.2 for 2 to 15 minutes at 37° C.,and measuring the concentration of bound antibody at 575 nm in an ELISAmicrotiter plate reader.

The ability of the present siRNA to down-regulate Ang1, Ang2 or Tie2expression can also be evaluated in vitro by measuring tube formation bybovine retinal endothelial cells (BRECs), using techniques within theskill in the art. An inhibition of tube formation indicates adown-regulation of Ang1, Ang2 or Tie2 by the present siRNA.

A suitable BREC tube formation assay comprises culturing BRECs onfibronectin-coated dishes containing Dulbecco's modified Eagle's medium(DMEM) with 5.5 mM glucose, 10% platelet-derived horse serum (PDHS;Wheaton, Pipersville, Pa.), 50 mg/mL heparin, and 50 U/mL endothelialcell growth factor (Roche Molecular Biochemicals). BRECs suitable foruse in the tube-formation assay exhibit endothelial homogeneity byimmunoreactivity for factor VIII antigen, and remain morphologicallyunchanged under these conditions as confirmed by light microscopy.

The tube formation assay can be performed as described in King G L etal., J. Clin. Invest. 75:1028-1036 (1985) and Otani A et al., Circ. Res.82: 619-628 (1998), the entire disclosures of which are hereinincorporated by reference. Briefly, an 8:1:1 (400 microliter) mixture ofVitrogen 100 (Celtrix, Palo Alto, Calif.), 0.2 N NaOH and 200 mM HEPESin 10× RPMI medium (Gibco BRL, Gaithersburg, Md.), containing 5microgram/mL fibronectin and 5 microgram/mL laminin, is added to 24-wellplates. After polymerization of the gels, 1.0×10⁵ of the cultured BRECsare seeded in the wells and incubated for 24 hours at 37° C. with DMEMcontaining 20% PDHS. The cell number is chosen to optimize the shape andtube length, as is known in the art (see King G L et al., 1985, supraand Otani A et al., 1998, supra). The medium is then removed, andadditional collagen gel is introduced onto the cell layer. Before makingthe collagen gel, reference points can be randomly marked in the centerarea of the bottom of each well, in order to measure the density persurface area of any tubelike structures formed by the BRECs. Either VEGFor hypoxia-conditioned medium is then added to the wells to induce tubeformation. One or more siRNA of the invention are then introduced intothe BRECs of certain wells by any suitable procedure (see below). Otherwells are treated with either no siRNA or a non-specific siRNA ascontrols. Inhibition of tube formation in the wells treated with siRNAas compared to the control wells indicates that expression of the targetRNA has been has been inhibited.

RNAi-mediated degradation of Ang1, Ang2 or Tie2 mRNA by an siRNA of theinvention can also be evaluated with animal models ofneovascularization, such as the retinopathy of prematurity (“ROP”) orchoroidal neovascularization (“CNV”) rat or mouse models. For example,areas of neovascularization in a CNV rat or mouse can be measured beforeand after administration of the present siRNA, as in Example 6 below. Areduction in the areas of neovascularization upon administration of thesiRNA indicates the down-regulation of target mRNA and an inhibition ofangiogenesis. Down-regulation of target mRNA and an inhibition ofangiogenesis is also demonstrated below in the streptozotocin-induceddiabetic retinopathy rat model (Example 3), a rat model of VEGF-inducedretinal vascular permeability and leukostasis (Example 4), and a ratmodel of ocular neovascularization induced by corneal/limbal injury(Example 5).

The mouse model of ischemia-induced retinal neovascularization asdescribed in Takagi et al., 2003, supra can also be used to detectRNAi-mediated degradation of Ang1, Ang2 or Tie2 with the present siRNA.Briefly, litters of 7-day-old (“postnatal day 7” or “P7”) C57BL/6J miceare exposed to 75%±2% oxygen for 5 days, and are then returned to roomair at P12 to produce retinal neovascularization. Mice of the same age,maintained in room air, serve as a control. Maximal retinalneovascularization is typically observed at P17, S days after return toroom air. One or more siRNA of the invention are injected subretinallyinto one eye of each treatment animal on P12 and P14. Either no siRNA,or a non-specific siRNA is injected into the contralateral eye as acontrol. At P17, the mice are killed by cardiac perfusion of 1 mL 4%paraformaldehyde in PBS, and the eyes are enucleated and fixed in 4%paraformaldehyde overnight at 4° C. before paraffin embedding. Serialsections of the paraffin-embedded eyes can be obtained for observationof the extent of neovascularization in the retina. Reducedneovascularization in the retinas of eyes treated with one or more siRNAof the invention, as compared to controls, indicate inhibition inexpression of the target mRNA.

As discussed above, the siRNA of the invention target can cause theRNAi-mediated degradation of Ang1, Ang2 or Tie2 mRNA, or alternativesplice forms, mutants or cognates thereof. Degradation of the targetmRNA by the present siRNA reduces the production of a functional geneproduct from the Ang1, Ang2 and/or Tie2 genes. Thus, the inventionprovides a method of inhibiting expression of Ang1, Ang2 or Tie2 in asubject, comprising administering an effective amount of an siRNA of theinvention to the subject, such that the target mRNA is degraded. As theproducts of the Ang1, Ang2 or Tie2 genes are involved in angiogenesis,the invention also provides a method of inhibiting angiogenesis in asubject by the RNAi-mediated degradation of the target mRNA by thepresent siRNA.

In the practice of the present methods, two or more siRNA comprisingdifferent target sequences in the Ang1, Ang2 or Tie2 mRNA can beadministered to the subject. Likewise, two or more siRNA, eachcomprising target sequences from a different target mRNA (i.e., Ang1,Ang2 and Tie2 mRNA) can also be administered to a subject.

As discussed above, Ang1 or Ang2 in conjunction with Tie2 appear topromote angiogenesis in the presence of VEGF, and Ang2 in conjunctionwith Tie2 appears to promote angiogenesis under hypoxic conditions.However, it is not clear whether VEGF and/or hypoxic conditions arerequired for Ang1 Ang2- and Tie2-mediated angiogenesis. Also,downregulation of either Ang1, Ang2 or Tie2 expression alone can besufficient to inhibit angiogenesis. It is therefore not necessary toverify the presence of VEGF or hypoxia in the practice of the presentmethods.

As used herein, a “subject” includes a human being or non-human animal.Preferably, the subject is a human being.

As used herein, an “effective amount” of the siRNA is an amountsufficient to cause RNAi-mediated degradation of the target mRNA, or anamount sufficient to inhibit the progression of angiogenesis in asubject.

RNAi-mediated degradation of the target mRNA can be detected bymeasuring levels of the target mRNA or protein in the cells of asubject, using standard techniques for isolating and quantifying mRNA orprotein as described above.

Inhibition of angiogenesis can be evaluated by directly measuring theprogress of pathogenic or nonpathogenic angiogenesis in a subject; forexample, by observing the size of a neovascularized area before andafter treatment with the siRNA of the invention. An inhibition ofangiogenesis is indicated if the size of the neovascularized area staysthe same or is reduced. Techniques for observing and measuring the sizeof neovascularized areas in a subject are within the skill in the art;for example, areas of choroid neovascularization can be observed byophthalmoscopy.

Inhibition of angiogenesis can also be inferred through observing achange or reversal in a pathogenic condition associated with theangiogenesis. For example, in AMD, a slowing, halting or reversal ofvision loss indicates an inhibition of angiogenesis in the choroid. Fortumors, a slowing, halting or reversal of tumor growth, or a slowing orhalting of tumor metastasis, indicates an inhibition of angiogenesis ator near the tumor site. Inhibition of non-pathogenic angiogenesis canalso be inferred from, for example, fat loss or a reduction incholesterol levels upon administration of the siRNA of the invention.

It is understood that the siRNA of the invention can mediate RNAinterference (and thus inhibit angiogenesis) in substoichiometricamounts. Without wishing to be bound by any theory, it is believed thatthe siRNA of the invention induces the RISC to degrade the target mRNAin a catalytic manner. Thus, compared to standard therapies for celladhesion or cell adhesion mediated pathologies, significantly less siRNAneeds to be administered to the subject to have a therapeutic effect.

One skilled in the art can readily determine an effective amount of thesiRNA of the invention to be administered to a given subject, by takinginto account factors such as the size and weight of the subject; theextent of the neovascularization or disease penetration; the age, healthand sex of the subject; the route of administration; and whether theadministration is regional or systemic. Generally, an effective amountof the siRNA of the invention comprises an intercellular concentrationat or near the neovascularization site of from about 1 nanomolar (nM) toabout 100 nM, preferably from about 2 nM to about 50 nM, more preferablyfrom about 2.5 nM to about 10 nM. Particularly preferred effectiveamounts of the siRNA of the invention can comprise an intercellularconcentration at or near the neovascularization site of about 1 nM,about 5 nM, or about 25 nM. It is contemplated that greater or lessereffective amounts of siRNA can be administered.

The present methods can be used to inhibit angiogenesis which isnon-pathogenic; i.e., angiogenesis which results from normal processesin the subject. Examples of non-pathogenic angiogenesis includeendometrial neovascularization, and processes involved in the productionof fatty tissues or cholesterol. Thus, the invention provides a methodfor inhibiting non-pathogenic angiogenesis; e.g., for controlling weightor promoting fat loss, for reducing cholesterol levels, an inhibitor ofthe menstrual cycle, or as an abortifacient.

The present methods can also inhibit angiogenesis which is associatedwith an angiogenic disease; i.e., a disease in which pathogenicity isassociated with inappropriate or uncontrolled angiogenesis. For example,most cancerous solid tumors generate an adequate blood supply forthemselves by inducing angiogenesis in and around the tumor site. Thistumor-induced angiogenesis is often required for tumor growth, and alsoallows metastatic cells to enter the bloodstream.

Other angiogenic diseases include AMD, psoriasis, rheumatoid arthritisand other inflammatory diseases. These diseases are characterized by thedestruction of normal tissue by newly formed blood vessels in the areaof neovascularization. For example, in the wet form of AMD, the choroidis invaded and destroyed by capillaries. The angiogenesis-drivendestruction of the choroid in AMD eventually leads to partial or fullblindness.

In another embodiment, the invention provides a method of treating asubject for complications arising from type I diabetes, by theRNAi-mediated degradation of the target mRNA by the present siRNA.Preferably, the complications arising from type I diabetes to be treatedby the present method are diabetic retinopathy, diabetic neuropathy,diabetic nephropathy, and macrovascular disease (including coronaryartery disease, cerebrovascular disease, and peripheral vasculardisease).

Preferably, an siRNA of the invention is used to inhibit the growth ormetastasis of solid tumors associated with cancers; for example breastcancer, lung cancer, head and neck cancer, brain cancer, abdominalcancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, liver cancer, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma; skin cancer(e.g., melanoma), lymphomas and blood cancer.

More preferably, an siRNA of the invention is used to treatcomplications arising from type I diabetes, such as diabeticretinopathy, diabetic neuropathy, diabetic nephropathy and macrovasculardisease.

Particularly preferably, an siRNA of the invention is used to inhibitocular neovascularization, for example to inhibit choroidalneovascularization in AMD.

For treating angiogenic diseases, the siRNA of the invention canadministered to a subject in combination with a pharmaceutical agentwhich is different from the present siRNA. Alternatively, the siRNA ofthe invention can be administered to a subject in combination withanother therapeutic method designed to treat the angiogenic disease. Forexample, the siRNA of the invention can be administered in combinationwith therapeutic methods currently employed for treating cancer orpreventing tumor metastasis (e.g., radiation therapy, chemotherapy, andsurgery). For treating tumors, the siRNA of the invention is preferablyadministered to a subject in combination with radiation therapy, or incombination with chemotherapeutic agents such as cisplatin, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

In the present methods, the present siRNA can be administered to thesubject either as naked siRNA, in conjunction with a delivery reagent,or as a recombinant plasmid or viral vector which expresses the siRNA.

Suitable delivery reagents for administration in conjunction with thepresent siRNA include the Mirus Transit TKO lipophilic reagent;lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), or liposomes. A preferred delivery reagent is a liposome.

Liposomes can aid in the delivery of the siRNA to a particular tissue,such as retinal or tumor tissue, and can also increase the bloodhalf-life of the siRNA. Liposomes suitable for use in the invention areformed from standard vesicle-forming lipids, which generally includeneutral or negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of factors such as the desired liposome size and half-lifeof the liposomes in the blood stream. A variety of methods are known forpreparing liposomes, for example as described in Szoka et al. (1980),Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which areherein incorporated by reference.

Preferably, liposomes encapsulating the present siRNA comprise a ligandmolecule that can target the liposome to cells such as endothelial cellswhich express Ang1, Ang2 or Tie2 at or near the site of angiogenesis.Ligands which bind to receptors prevalent in vascular EC, such asmonoclonal antibodies that bind to EC surface antigens, are preferred.

Particularly preferably, the liposomes encapsulating the present siRNA'sare modified so as to avoid clearance by the mononuclear macrophage andreticuloendothelial systems, for example by havingopsonization-inhibition moieties bound to the surface of the structure.In one embodiment, a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer which significantly decreases the uptakeof the liposomes by the macrophage-monocyte system (“MMS”) andreticuloendothelial system (“RES”); e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is herein incorporated byreference. Liposomes modified with opsonization-inhibition moieties thusremain in the circulation much longer than unmodified liposomes. Forthis reason, such liposomes are sometimes called “stealth” liposomes.

Stealth liposomes are known to accumulate in tissues fed by porous or“leaky” microvasculature. Thus, tissue characterized by suchmicrovasculature defects, for example solid tumors, will efficientlyaccumulate these liposomes; see Gabizon, et al. (1988), P.N.A.S., USA,18: 6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation inthe liver and spleen. Thus, liposomes of the invention that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe present siRNA to tumor cells.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM₁. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginicacid, carrageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups.

Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Recombinant plasmids which express siRNA of the invention are discussedabove. Such recombinant plasmids can also be administered directly or inconjunction with a suitable delivery reagent, including the MinisTransit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin;polycations (e.g., polylysine) or liposomes. Recombinant viral vectorswhich express siRNA of the invention are also discussed above, andmethods for delivering such vectors to cells of a subject which areexpressing Ang1, Ang2 or Tie2 are within the skill in the art.

The siRNA of the invention can be administered to the subject by anymeans suitable for delivering the siRNA to the cells expressing Ang1,Ang2 or Tie2. For example, the siRNA can be administered by gene gun,electroporation, or by other suitable parenteral or enteraladministration routes.

Suitable enteral administration routes include oral, rectal, orintranasal delivery.

Suitable parenteral administration routes include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissueadministration (e.g., peri-tumoral and intra-tumoral injection,intra-retinal injection or subretinal injection); subcutaneous injectionor deposition including subcutaneous infusion (such as by osmoticpumps); direct (e.g., topical) application to the area at or near thesite of neovascularization, for example by a catheter or other placementdevice (e.g., a corneal pellet or a suppository, eye-dropper, or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Suitable placement devices include the ocular implantsdescribed in U.S. Pat. Nos. 5,902,598 and 6,375,972, and thebiodegradable ocular implants described in U.S. Pat. No 6,331,313, theentire disclosures of which are herein incorporated by reference. Suchocular implants are available from Control Delivery Systems, Inc.(Watertown, Mass.) and Oculex Pharmaceuticals, Inc. (Sunnyvale, Calif.).

In a preferred embodiment, injections or infusions of the siRNA aregiven at or near the site of neovascularization. For example, the siRNAof the invention can be delivered to retinal pigment epithelial cells inthe eye. Preferably, the siRNA is administered topically to the eye,e.g. in liquid or gel form to the lower eye lid or conjunctivalcul-de-sac, or by electroporation or iontophoresis, as is within theskill in the art (see, e.g., Acheampong A A et al, 2002, Drug Metabol.and Disposition 30: 421-429, the entire disclosure of which is hereinincorporated by reference).

Typically, the siRNA of the invention is administered topically to theeye in volumes of from about 5 microliters to about 75 microliters, forexample from about 7 microliters to about 50 microliters, preferablyfrom about 10 microliters to about 30 microliters. The siRNA of theinvention is highly soluble in aqueous solutions, and it is understoodthat topical instillation in the eye of siRNA in volumes greater than 75microliters can result in loss of siRNA from the eye through spillageand drainage. Thus, it is preferable to administer a high concentrationof siRNA (e.g., about 10 to about 200 mg/ml, or about 100 to about 1000nM) by topical instillation to the eye in volumes of from about 5microliters to about 75 microliters.

A particularly preferred parenteral administration route is intraocularadministration. It is understood that intraocular administration of thepresent siRNA can be accomplished by injection or direct (e.g., topical)administration to the eye, as long as the administration route allowsthe siRNA to enter the eye. In addition to the topical routes ofadministration to the eye described above, suitable intraocular routesof administration include intravitreal, intraretinal, subretinal,subtenon, peri- and retro-orbital, trans-corneal and trans-scleraladministration. Such intraocular administration routes are within theskill in the art; see, e.g., and Acheampong AA et al, 2002, supra; andBennett et al. (1996), Hum. Gene Ther. 7: 1763-1769 and Ambati J et al.,2002, Progress in Retinal and Eye Res. 21: 145-151, the entiredisclosures of which are herein incorporated by reference.

The siRNA of the invention can be administered in a single dose or inmultiple doses. Where the administration of the siRNA of the inventionis by infusion, the infusion can be a single sustained dose or can bedelivered by multiple infusions.

One skilled in the art can also readily determine an appropriate dosageregimen for administering the siRNA of the invention to a given subject.For example, the siRNA can be administered to the subject once, such asby a single injection or deposition at or near the neovascularizationsite. Alternatively, the siRNA can be administered to a subject multipletimes daily or weekly. For example, the siRNA can be administered to asubject once weekly for a period of from about three to abouttwenty-eight weeks, more preferably from about seven to about ten weeks.In a preferred dosage regimen, the siRNA is injected at or near the siteof neovascularization (e.g., intravitreally) once a week for sevenweeks. It is understood that periodic administrations of the siRNA ofthe invention for an indefinite length of time may be necessary forsubjects suffering from a chronic neovascularization disease, such aswet AMD or diabetic retinopathy.

Where a dosage regimen comprises multiple administrations or theadministration of two or more siRNA, each of which comprise a differenttarget sequence, it is understood that the effective amount of siRNAadministered to the subject can comprise the total amount of siRNAadministered over the entire dosage regimen.

The siRNA of the invention are preferably formulated as pharmaceuticalcompositions prior to administering to a subject, according totechniques known in the art. Pharmaceutical compositions of the presentinvention are characterized as being at least sterile and pyrogen-free.As used herein, “pharmaceutical formulations” include formulations forhuman and veterinary use. Methods for preparing pharmaceuticalcompositions of the invention are within the skill in the art, forexample as described in Remington's Pharmaceutical Science, 17th ed.,Mack Publishing Company, Easton, Pa. (1985), the entire disclosure ofwhich is herein incorporated by reference.

The present pharmaceutical formulations comprise an siRNA of theinvention (e.g., 0.1 to 90% by weight), or a physiologically acceptablesalt thereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and thelike.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

For topical administration to the eye, conventional intraocular deliveryreagents can be used. For example, pharmaceutical compositions of theinvention for topical intraocular delivery can comprise saline solutionsas described above, corneal penetration enhancers, insoluble particles,petrolatum or other gel-based ointments, polymers which undergo aviscosity increase upon instillation in the eye, or mucoadhesivepolymers. Preferably, the intraocular delivery reagent increases cornealpenetration, or prolongs preocular retention of the siRNA throughviscosity effects or by establishing physicochemical interactions withthe mucin layer covering the corneal epithelium.

Suitable insoluble particles for topical intraocular delivery includethe calcium phosphate particles described in U.S. Pat. No. 6,355,271 ofBell et al., the entire disclosure of which is herein incorporated byreference. Suitable polymers which undergo a viscosity increase uponinstillation in the eye include polyethylenepolyoxypropylene blockcopolymers such as poloxamer 407 (e.g., at a concentration of 25%),cellulose acetophthalate (e.g., at a concentration of 30%), or alow-acetyl gellan gum such as Gelrite® (available from CP Kelco,Wilmington, Del.). Suitable mucoadhesive polymers include hydrocolloidswith multiple hydrophilic functional groups such as carboxyl, hydroxyl,amide and/or sulfate groups; for example, hydroxypropylcellulose,polyacrylic acid, high-molecular weight polyethylene glycols(e.g., >200,000 number average molecular weight), dextrans, hyaluronicacid, polygalacturonic acid, and xylocan. Suitable corneal penetrationenhancers include cyclodextrins, benzalkonium chloride, polyoxyethyleneglycol lauryl ether (e.g., Brij® 35), polyoxyethylene glycol stearylether (e.g., Brij® 78), polyoxyethylene glycol oleyl ether (e.g., Brij®98), ethylene diamine tetraacetic acid (EDTA), digitonin, sodiumtaurocholate, saponins and polyoxyethylated castor oil such as CremaphorEL.

For solid compositions, conventional nontoxic solid carriers can beused; for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of one or more siRNA of the invention. Apharmaceutical composition for aerosol (inhalational) administration cancomprise 0.01-20% by weight, preferably 1%-10% by weight, of one or moresiRNA of the invention encapsulated in a liposome as described above,and propellant. A carrier can also be included as desired; e.g.,lecithin for intranasal delivery.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLE 1 Inhibition of Ang2 Expression in Cultured Human Cells withsiRNA Targeted to Ang2 mRNA

Human embryonic kidney (HEK-293 cells) were cultured in 24 well platesat 37° C. with 5% CO₂ overnight in standard growth medium. Transfectionswere performed the next day when the cells were about 70% confluent. TheHEK-293 cells were separately transfected with twelve different siRNA(25 nM each) targeted to human Ang2 (“hANG2”) mRNA, mixed with a CaPitransfection reagent. These twelve siRNAs target the sequences listed inTable 1, and all siRNAs contained 3′ TT overhangs on each strand.Control cells were transfected with CaPi transfection reagent lackingsiRNA, or a nonspecific siRNA targeted to enhanced green fluorescentprotein (EGFP siRNA) mixed with CaPi transfection reagent. Forty eighthours post-transfection, the growth medium was removed from all wells,and a human ANG2 ELISA (R & D systems, Minneapolis, Minn.) was performedas described in the Quantikine human ANG2 ELISA protocol, the entiredisclosure of which is herein incorporated by reference. ELISA resultswere read on an AD340 plate reader (Beckman Coulter), and are reportedin FIG. 1.

TABLE 1  Target Sequences for hANG2 siRNAs Tested in HEK-293 CellsTarget Sequence SEQ ID NO: siRNA AAGAGCATGGACAGCATAGGA 232 hANG2#1AACCAGACGGCTGTGATGATA 254 hANG2#2 AAACGCGGAAGTTAACTGATG 262 hANG2#3AACGCGGAAGTTAACTGATGT 263 hANG2#4 AAGAAGGTGCTAGCTATGGAA 291 hANG2#5AATAGTGACTGCCACGGTGAA 316 hANG2#6 AATAACTTACTGACTATGATG 323 hANG2#7AATCAGGACACACCACAAATG 336 hANG2#8 AAATGGCATCTACACGTTAAC 337 hANG2#9AATGGCATCTACACGTTAACA 338  hANG2#10 AATTATTCAGCGACGTGAGGA 344  hANG2#11AAGAACTCAATTATAGGATTC 366  hANG2#12

As can be seen from FIG. 1, the level of hANG2 protein secreted into thegrowth medium was reduced in HEK-293 cells transfected with hANG2#2, #3,#4, #9 and #10 siRNA. Transfection of HEK-293 cells with non-specificsiRNA had no apparent effect on hANG2 protein levels.

After the growth medium was removed from each well, a cytotoxicity assaywas performed on the cells as follows. Complete growth medium containing10% AlamarBlue (Biosource, Camarillo, Calif.) was added to each well,and cells were incubated at 37° C. with 5% CO₂ for 3 hours. Cellproliferation was measured by detecting the color change of mediumcontaining AlamarBlue which resulted from cell metabolic activity. Thecytotoxicity assay results were read on an AD340 plate reader (BeckmanCoulter), and are reported in FIG. 2.

As can be seen in FIG. 2, the transfection of HEK-293 cells with thehANG2#8 and #12 siRNA produced a slight reduction in cell growth ascompared to control cells. The remaining hANG2 siRNAs showed no apparentcytotoxicity as compared with control cells.

After cytotoxicity assay was performed, the AlamarBlue-containing mediumin each well was completely removed and RNA extractions were performedusing the RNAqueous RNA isolation kit (Ambion, Austin, Tex.). The levelsof hANG2 mRNA in the HEK-293 cells were measured by a quantitativereverse-transcriptase/polymerase chain reaction (RT-PCR) assay.Expression of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)mRNA was used as a internal control. The levels of hANG2 mRNA in HEK-293cells were reduced by transfection with the hANG2 siRNA compared tocontrol cells, in a pattern which correlated with the reduction in hANG2protein shown in FIG. 1.

EXAMPLE 2 Dose-Response of hAng2#2 and #3 in Cultured Human Cells

HEK-293 cells were grown to about 70% confluency as in Example 1 above.The cells were then transfected with I nanomolar (“nM”), 5 nM or 25 nMdoses of hANG2#2 or #3 siRNA in CaPi transfection reagent. Control cellswere transfected with 25 nM nonspecific EGFP siRNA in CaPi transfectionreagent, or with transfection reagent alone. hANG2 protein levels weremeasured in the growth medium at 48 hours post-transfection by ANG2ELISA as described in Example 1 above, and the results are presented inFIG. 3.

As can be seen from FIG. 3, the levels of hANG2 protein level werereduced in HEK-293 cells transfected with the hANG2#2 and #3 siRNA, in adose-dependent manner. All doses of hANG2#2 siRNA and the 5 and 25 nMdoses of hANG2#3 siRNA reduced the level of hANG2 protein secreted intothe growth medium, as compared to control cells. The 1 nM dose ofhANG2#3 siRNA did not reduce the hANG2 protein level as compared tocontrol cells mock-transfected with transfection reagent alone. However,the level of hANG2 protein secreted by cells transfected with 1 nMhANG2#3 siRNA was slightly reduced as compared to control cellstransfected with the nonspecific siRNA. Transfections with thenon-specific siRNA had no apparent effect on hANG2 protein levels.

A cytotoxicity assay was performed on the control HEK-293 cells and theHEK-293 cells transfected with the different doses of hANG2#2 and #3siRNA as described above in Example 1. As can be seen in FIG. 4, thetransfection of HEK-293 cells with 5 nM hANG2#2 siRNA produced a slightreduction in cell growth as compared to control cells mock-transfectedwith transfection reagent alone. There was no apparent toxicity of the 5nM hANG2#2 siRNA dose as compared to control cells transfected with thenonspecific siRNA. The remaining doses of hANG2#2 or #3 siRNA showed noapparent cytotoxicity as compared with control cells transfected withnonspecific siRNA or with transfection reagent alone.

EXAMPLE 3 Treatment of Streptozotocin-Induced Diabetic Retinopathy withsiRNA Targeted to Ang1, Ang2 or Tie2

Vascular leakage and non-perfusion in the retinas of individuals withdiabetic retinopathy is spatially and temporally associated withleukocyte stasis. See, e.g., Miyamoto K et al. (1999), Proc. Nat. Acad.Sci. USA 96(19):10836-41, the entire disclosure of which is hereinincorporated by reference. It is expected that intravitreal injection ofsiRNA targeted to Ang1, Ang2 or Tie2 will decrease leukocyte stasis, andtherefore reduce retinal vascular permeability, in diabetic rats.

Long-Evans rats (approximately 200 g) will be injected withstreptozotocin in citrate buffer intravenously after an overnight fastto induce diabetes, as described in Miyamoto K et al. (1999), supra.Long-Evans rats (approximately 200 g) will be injected with citratebuffer alone after an overnight fast as a control. The serum blood-sugarwill be measured and blood pressure will be recorded daily. Elevatedlevels of serum blood sugar as compared to control animals areconsidered diabetic.

Intravitreal injections of siRNA targeted to Ang1, Ang2 or Tie2(“experimental siRNA”) will be performed OD in each rat. Non-specificsiRNA will be injected as a control OS. The overall group scheme will beas shown in Table 2.

TABLE 2 Overall Group Scheme OD OS (experimental siRNA) (non-specificsiRNA) Diabetic Rat (STZ) Experimental group Control Non-diabetic RatControl Control

At day 7 post treatment, the rats will be subjected to Acridine OrangeLeukocyte Fluorography (AOLF), as described in Miyamoto K et al (1999),supra. Briefly, the rats will be anaesthetized, and their pupils dilatedwith tropicamide. The rats will then be injected intravenously withacridine orange suspended in sterile saline. The fundus of each eye willbe observed and imaged with a scanning laser ophthalmoscope (argon bluelaser as a light source) for leukocyte stasis. The rats will then beperfused with fluorescein dextran and the eyes will be further imaged.The density of leukocyte stasis will be calculated as a percentage ofbright pixels in a 10 disk diameter radius. The density of leukocytestasis will be used as an endpoint.

Also on day 7, the rats will undergo an isotope dilution technique toquantify vascular leakage, as described in Miyamoto K et al (1999),supra. Briefly, the rats will be injected intravenously with I¹²⁵ in BSAat one time point, and with I¹³¹ at a second time point. The rats willbe sacrificed minutes after the second injection, the retinas will beisolated, and arterial samples will be taken. The retinas and thearterial samples will be analyzed using γ-spectroscopy after correctingfor activity in the retinas using a quantitative index of iodineclearance. The measurements will then be normalized for exact dosegiven, body weight and tissue weight. The corrected quantity of γactivity will be used as a marker of vascular leakage in the retina(second endpoint). It is expected that the γ activity will be decreasedin the retinas of the experimental animals, indicating decreasedvascular leakage.

EXAMPLE 4 Treatment of VEGF-Induced Vascular Permeability andLeukostasis with siRNA Targeted to Ang1, Ang2 or Tie2

The presence of VEGF in the eye causes retinal leukostasis thatcorresponds with increased vascular permeability and capillarynon-perfusion in the retina. See, e.g., Miyamoto K et al. (2000), Am. J.Pathol. 156(5):1733-9, the entire disclosure of which is hereinincorporated by reference. It is expected that intravitreal injection ofsiRNA targeted to Ang1, Ang2 or Tie2 will decrease the permeability andleukostasis created by intravitreal injection of VEGF in rats.

Long-Evans rats (approximately 200 g) will be anaesthetized and injectedintravitreally with VEGF in buffer OU. siRNA targeted to Ang1, Ang2 orTie2 (“experimental siRNA”) will be simultaneously delivered OD to eachrat by intravitreal injection. Non-specific siRNA will be injectedintravitreally as a control OS. Additional controls will include ratsinjected with buffer alone (no VEGF). The overall group scheme will beas shown in Table 3.

TABLE 3 Overall Group Scheme OD OS (experimental siRNA) (Non-specificsiRNA) VEGF Experimental group Control Buffer Control Control

At 24 hours post injection the rats are subjected to AOLF and an isotopedilution technique as described in Example 3.

EXAMPLE 5 Treatment of Neovascularization in Eyes Subjected toCorneal/Limbal Injury with siRNA Targeted to Ang1, Ang2 or Tie2

Injury to the ocular surface can cause the destruction of corneal limbalstem cells. Destruction of these cells induces a VEGF-dependent cornealneovascularization, which can lead to blindness. The VEGF which drivesthe neovascularization is supplied by neutrophils and monocytes thatinfiltrate the cornea after injury to the ocular surface. See, e.g.,Moromizato Y et al. (2000), Am. J. Pathol. 157(4):1277-81, the entiredisclosure of which is herein incorporated by reference in its entirety.It is expected that siRNA targeted to Ang1, Ang2 or Tie2 applied to thecornea after limbal injury will decrease the resultant area ofneovascularization of the cornea in mice. The area of neovascularizationcan be measured directly. Alternatively, a reduction in cornealneovascularization can be inferred from a decrease in the number ofVEGF-producing polymorphonuclear cells in the cornea.

Corneal neovascularization will be induced in C57Bl/6 by damaging thelimbus, as described in Moromizato Y et al., supra. Briefly, the micewill be anaesthetized and sodium hydroxide will be applied to thecornea. The corneal and limbal epithelia will be debrided using acorneal knife OU. siRNA targeted to Ang1, Ang2 or Tie2 will be appliedto the corneal surface OD immediately, after removal, and 3 times a dayfor the duration of the study (7 days). Non-specific siRNA will beadministered OS with the same dosing regimen as a control.

On days 2, 4 and 7 after debridement of the corneal and limbalepithelia, mice will be evaluated for the degree of cornealneovascularization as described in Moromizato Y et al., supra. Briefly,endothelial-specific, fluorescein-conjugated lectin will be injectedintravenously. Thirty minutes after injection, mice will be sacrificed,and the eyes will be harvested and fixed in formalin for 24 hours. Flatmounts of the corneas will be made, and pictures of the corneal flatmounts will be taken under fluorescent microscopy and imported intoOpenlab software for analysis. Using the Openlab software, thresholdlevel of fluorescence will be set, above which only vessels are seen.The area of fluorescent vessels and the area of the cornea (demarcatedby the limbal arcade) will be calculated. The area of vessels will bedivided by the total corneal area, and this value will equal the percentneovascular area. The percent neovascular area of the treatment andcontrol groups will be compared.

On days 2, 4 and 7 after debridement of the corneal and limbalepithelia, additional mice will be sacrificed for quantification ofcorneal polymorphonuclear cells (PMNs) as described in Moromizato Y etal., supra. Briefly, mice will be sacrificed, and the eyes will beharvested and fixed in formalin for 24 hours. After formalin fixation,the enucleated eyes will be embedded in paraffin and sectioned. Oneparaffin section from each eye which correlates to the cornealanatomical center will be chosen and used for microscopy. The PMNs(identified as multilobulated cells) will be counted on this onesection, and the number of PMNs in the sections from the treatment andcontrol groups will be compared.

EXAMPLE 6 Treatment of Laser-Induced Choroidal Neovascularization withsiRNA Targeted to Ang1, Ang2 or Tie2

Laser photocoagulation that ruptures Bruch's membrane will inducechoroidal neovascularization (CNV) similar to that seen in wet maculardegeneration. It is expected that intravitreal injection of siRNAtargeted to Ang1, Ang2 or Tie2 will decrease the area of laser-inducedCNV in mice.

CNV will be induced in mice by the procedure described in Sakurai E etal. (2003), Invest. Ophthalmol. & Visual Sci. 44(61:2743-9, the entiredisclosure of which is herein incorporated by reference. Briefly,C57Bl/6 mice will be anaesthetized, and their pupils will be dilatedwith tropicamide. The retinas of the mice will be laser photocoagulatedwith one laser spot at the 9, 12, and 3 o'clock positions of eachretinal OU. Immediately following laser photocoagulation, inject siRNAtargeted to Ang1, Ang2 or Tie2 will be injected intravitreally OD.Non-specific siRNA will be injected intravitreally OS as a control.

Fourteen days after laser photocoagulation, the mice will be sacrificedand retinal flat mounts will be prepared for CNV area quantification asdescribed in Sakurai E et al. (2003), supra. Briefly, the mice will beanaesthetized, the chest will be opened, and the descending aorta willbe cross-clamped. The right atrium will then be clipped andfluorescein-labeled dextran will be injected slowly into the leftventricle.

After injection of the fluorescein-labeled dextran, the eyes will beenucleated and fixed in paraformaldehyde for 24 hours. The anteriorchamber and retina will then be removed, and a flat mount of eachchoroid will be prepared for analysis. Choroidal flat mounts will beanalyzed by taking a picture of each under fluorescent microscopy, andimporting the picture into Openlab software. Using the Openlab software,the area of neovascularization will be outlined and quantified, beingsure known laser location is compared to the fluorescent tuft. Theneovascular area of the treatment animals will be compared to that ofthe control animals.

We claim:
 1. An isolated siRNA comprising a sense RNA strand and anantisense RNA strand, wherein the sense and an antisense RNA strandsform an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence substantially identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2mRNA, or an alternative splice form, mutant or cognate thereof.
 2. ThesiRNA of claim 1, wherein the cognate of the human Ang1, Ang2 or Tie2mRNA sequence is SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:7.
 3. The siRNA of claim 1, wherein the sense RNA strand comprises oneRNA molecule, and the antisense RNA strand comprises one RNA molecule.4. The siRNA of claim 1, wherein the sense and antisense RNA strandsforming the RNA duplex are covalently linked by a single-strandedhairpin.
 5. The siRNA of claim 1, wherein the siRNA further comprisesnon-nucleotide material.
 6. The siRNA of claim 1, wherein the siRNAfurther comprises an addition, deletion, substitution or alteration ofone or more nucleotides.
 7. The siRNA of claim 1, wherein the sense andantisense RNA strands are stabilized against nuclease degradation. 8.The siRNA of claim 1, further comprising a 3′ overhang.
 9. The siRNA ofclaim 8, wherein the 3′ overhang comprises from 1 to about 6nucleotides.
 10. The siRNA of claim 8, wherein the 3′ overhang comprisesabout 2 nucleotides.
 11. The siRNA of claim 3 wherein the sense RNAstrand comprises a first 3′ overhang, and the antisense RNA strandcomprises a second 3′ overhang.
 12. The siRNA of claim 11, wherein thefirst and second 3′ overhangs separately comprise from 1 to about 6nucleotides.
 13. The siRNA of claim 12, wherein the first 3′ overhangcomprises a dinucleotide and the second 3′ overhang comprises adinucleotide.
 14. The siRNA of claim 13, where the dinucleotidecomprising the first and second 3′ overhangs is dithymidylic acid (TT)or diuridylic acid (uu).
 15. The siRNA of claim 8, wherein the 3′overhang is stabilized against nuclease degradation.
 16. A recombinantplasmid comprising nucleic acid sequences for expressing an siRNAcomprising a sense RNA strand and an antisense RNA strand, wherein thesense and an antisense RNA strands form an RNA duplex, and wherein thesense RNA strand comprises a nucleotide sequence substantially identicalto a target sequence of about 19 to about 25 contiguous nucleotides inhuman Ang1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant orcognate thereof.
 17. The recombinant plasmid of claim 16, wherein thenucleic acid sequences for expressing the siRNA comprise an inducible orregulatable promoter.
 18. The recombinant plasmid of claim 16, whereinthe nucleic acid sequences for expressing the siRNA comprise a sense RNAstrand coding sequence in operable connection with a polyT terminationsequence under the control of a human U6 RNA promoter, and an antisenseRNA strand coding sequence in operable connection with a polyTtermination sequence under the control of a human U6 RNA promoter. 19.The recombinant plasmid of claim 16, wherein the plasmid comprises a CMVpromoter.
 20. A recombinant viral vector comprising nucleic acidsequences for expressing an siRNA comprising a sense RNA strand and anantisense RNA strand, wherein the sense and an antisense RNA strandsform an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence substantially identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2mRNA, or an alternative splice form, mutant or cognate thereof.
 21. Therecombinant viral vector of claim 20, wherein the nucleic acid sequencesfor expressing the siRNA comprise an inducible or regulatable promoter.22. The recombinant viral vector of claim 20, wherein the nucleic acidsequences for expressing the siRNA comprise a sense RNA strand codingsequence in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter, and an antisense RNA strandcoding sequence in operable connection with a polyT termination sequenceunder the control of a human U6 RNA promoter.
 23. The recombinant viralvector of claim 20, wherein the recombinant viral vector is selectedfrom the group consisting of an adenoviral vector, an adeno-associatedviral vector, a lentiviral vector, a retroviral vector, and a herpesvirus vector.
 24. The recombinant viral vector of claim 20, wherein therecombinant viral vector is pseudotyped with surface proteins fromvesicular stomatitis virus, rabies virus, Ebola virus, or Mokola virus.25. The recombinant viral vector of claim 23, wherein the recombinantviral vector comprises an adeno-associated viral vector.
 26. Apharmaceutical composition comprising an siRNA and a pharmaceuticallyacceptable carrier, wherein the siRNA comprises a sense RNA strand andan antisense RNA strand, wherein the sense and an antisense RNA strandsform an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence substantially identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2mRNA, or an alternative splice form, mutant or cognate thereof.
 27. Thepharmaceutical composition of claim 26, further comprising lipofectin,lipofectamine, cellfectin, polycations, or liposomes.
 28. Apharmaceutical composition comprising the plasmid of claim 16, or aphysiologically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 29. The pharmaceutical composition of claim 28,further comprising lipofectin, lipofectamine, cellfectin, polycations,or liposomes.
 30. A pharmaceutical composition comprising the viralvector of claim 20 and a pharmaceutically acceptable carrier.
 31. Amethod of inhibiting expression of human Ang1, Ang2 or Tie2 mRNA, or analternative splice form, mutant or cognate thereof, comprisingadministering to a subject an effective amount of an siRNA comprising asense RNA strand and an antisense RNA strand, wherein the sense and anantisense RNA strands form an RNA duplex, and wherein the sense RNAstrand comprises a nucleotide sequence substantially identical to atarget sequence of about 19 to about 25 contiguous nucleotides in humanAng1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant orcognate thereof, such that the human Ang1, Ang2 or Tie2 mRNA, or analternative splice form, mutant or cognate thereof, is degraded.
 32. Themethod of claim 31, wherein the subject is a human being.
 33. The methodof claim 31, wherein the siRNA is administered in conjunction with adelivery reagent.
 34. The method of claim 33, wherein the delivery agentis selected from the group consisting of lipofectin, lipofectamine,cellfectin, polycations, and liposomes.
 35. The method of claim 34,wherein the delivery agent is a liposome.
 36. The method claim 35,wherein the liposome comprises a ligand which targets the liposome tocells expressing Ang1, Ang2 or Tie2.
 37. The method of claim 36, whereinthe cells are endothelial cells.
 38. The method of claim 37, wherein theligand comprises a monoclonal antibody.
 39. The method of claim 35,wherein the liposome is modified with an opsonization-inhibition moiety.40. The method of claim 39, wherein the opsonization-inhibiting moietycomprises a PEG, PPG, or derivatives thereof.
 41. The method of claim31, wherein the siRNA is expressed from a recombinant plasmid.
 42. Themethod of claim 31, wherein the siRNA is expressed from a recombinantviral vector.
 43. The method of claim 42, wherein the recombinant viralvector comprises an adenoviral vector, an adeno-associated viral vector,a lentiviral vector, a retroviral vector, or a herpes virus vector. 44.The method of claim 43, wherein the recombinant viral vector ispseudotyped with surface proteins from vesicular stomatitis virus,rabies virus, Ebola virus, or Mokola virus.
 45. The method of claim 42,wherein the recombinant viral vector comprises an adeno-associated viralvector.
 46. The method of claim 31, wherein two or more siRNA areadministered to the subject, and wherein each siRNA comprises anucleotide sequence which is substantially identical to a differentAng1, Ang2 or Tie2 mRNA target sequence.
 47. The method of claim 31,wherein two or more siRNA are administered to the subject, and whereineach siRNA administered comprises a nucleotide sequence which issubstantially identical to a target sequence from a different targetmRNA.
 48. The method of claim 31, wherein the siRNA is administered byan enteral administration route.
 49. The method of claim 48, wherein theenteral administration route is selected from the group consisting oforal, rectal, and intranasal.
 50. The method of claim 31, wherein thesiRNA is administered by a parenteral administration route.
 51. Themethod of claim 50, wherein the parenteral administration route isselected from the group consisting of intravascular administration,peri- and intra-tissue administration, subcutaneous injection ordeposition, subcutaneous infusion, intraocular administration, anddirect application at or near the site of neovascularization.
 52. Themethod of claim 51, wherein the intravascular administration is selectedfrom the group consisting of intravenous bolus injection, intravenousinfusion, intra-arterial bolus injection, intra-arterial infusion andcatheter instillation into the vasculature.
 53. The method of claim 51,wherein the peri- and intra-tissue injection is selected from the groupconsisting of peri-tumoral injection, intra-tumoral injection,intra-retinal injection, and subretinal injection.
 54. The method ofclaim 51, wherein the intraocular administration comprises intravitreal,intraretinal, subretinal, subtenon, peri- and retro-orbital,trans-corneal or trans-scleral administration.
 55. The method of claim51, wherein the direct application at or near the site ofneovascularization comprises application by catheter, corneal pellet,eye dropper, suppository, an implant comprising a porous material, animplant comprising a non-porous material, or an implant comprising agelatinous material.
 56. The method of claim 55, wherein the site ofneovascularization is in the eye, and the direct application at or nearthe site of neovascularization comprises application by eyedropper. 57.A method of inhibiting angiogenesis in a subject, comprisingadministering to a subject an effective amount of an siRNA comprising asense RNA strand and an antisense RNA strand, wherein the sense and anantisense RNA strands form an RNA duplex, and wherein the sense RNAstrand comprises a nucleotide sequence substantially identical to atarget sequence of about 19 to about 25 contiguous nucleotides in humanAng1, Ang2 or Tie2 mRNA, or an alternative splice form, mutant orcognate thereof.
 58. The method of claim 57, wherein the angiogenesis ispathogenic.
 59. The method of claim 57, wherein the angiogenesis isnon-pathogenic.
 60. The method of claim 59, wherein the non-pathogenicangiogenesis is associated with production of fatty tissues orcholesterol production.
 61. The method of claim 59, wherein thenon-pathogenic angiogenesis comprises endometrial neovascularization.62. A method of treating an angiogenic disease in a subject, comprisingadministering to a subject in need of such treatment an effective amountof an siRNA comprising a sense RNA strand and an antisense RNA strand,wherein the sense and an antisense RNA strands form an RNA duplex, andwherein the sense RNA strand comprises a nucleotide sequencesubstantially identical to a target sequence of about 19 to about 25contiguous nucleotides in human Ang1, Ang2 or Tie2 mRNA, or analternative splice form, mutant or cognate thereof, such thatangiogenesis associated with the angiogenic disease is inhibited. 63.The method of claim 62, wherein the angiogenic disease comprises a tumorassociated with a cancer.
 64. The method of claim 63, wherein the canceris selected from the group consisting of breast cancer, lung cancer,head and neck cancer, brain cancer, abdominal cancer, colon cancer,colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma,liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovariancancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm'stumor, multiple myeloma, skin cancer, lymphoma, and blood cancer. 65.The method of claim 62, wherein the angiogenic disease is selected fromthe group consisting of diabetic retinopathy and age-related maculardegeneration.
 66. The method of claim 65, wherein the angiogenic diseaseis age-related macular degeneration.
 67. The method of claim 62, whereinthe siRNA is administered in combination with a pharmaceutical agent fortreating angiogenic disease, which pharmaceutical agent is differentfrom the siRNA.
 68. The method of claim 67, wherein angiogenic diseaseis cancer, and the pharmaceutical agent comprises a chemotherapeuticagent.
 69. The method of claim 68, wherein the chemotherapeutic agent isselected from the group consisting of cisplatin, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin, andtamoxifen.
 70. The method of claim 62, wherein the siRNA is administeredto a subject in combination with another therapeutic method designed totreat the angiogenic disease.
 71. The method of claim 70, wherein theangiogenic disease is cancer, and the siRNA is administered incombination with radiation therapy, chemotherapy or surgery.
 72. Amethod of treating complications arising from type I diabetes in asubject, comprising administering to a subject in need of such treatmentan effective amount of an siRNA comprising a sense RNA strand and anantisense RNA strand, wherein the sense and an antisense RNA strandsfrom an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence substantially identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in human Ang1, Ang2 or Tie2mRNA, or an alternative splice form, mutant or cognate thereof.
 73. Themethod of claim 72, wherein the complications arising from type Idiabetes are selected from the group consisting of diabetic retinopathy,diabetic neuropathy, diabetic nephropathy and macrovascular disease. 74.The method of claim 73, wherein the macrovascular disease is coronaryartery disease, cerebrovascular disease or peripheral vascular disease.